It is known to form an immunological "sandwich" wherein an antibody is attached in a stable association with a solid surface, such as an absorbent membrane or microtitre well. The analyte is then free to interact with an antibody forming a high affinity complex. A second labeled antibody with specificities for the analyte is bound to this complex to form the immunological sandwich. This method of capturing analytes immunologically is frequently efficient in binding trace quantities in a background matrix. Further, this stable complex tends to allow extensive washing to remove interfering substances found in the matrix.
Certain analytes, such as C. trachomatis LPS, primarily because of their small size, do not lend themselves well to forming such an immunological sandwich. The epitope of such analytes is not sufficiently large (for example 3.5 kd in C. trachomatis) to easily bind the two antibodies. Consequently, a non-immunological form of analyte capture is often employed.
Other microbial LPS molecules of a much higher molecular weight that would be amenable to the typical sandwich assay might also be captured by a similar non-specific mechanism. Such non-specific forms of capture, because of much smaller analyte binding affinities, are not often as efficient in capturing sufficient analyte for detection especially when these analytes are present in low concentrations. The result is lower potential assay sensitivities. The antibody and enzyme conjugate reagents must be optimized in such a way as to enhance signal production. This often results in decreasing the specific signal with respect to background signal.
The prior art has attempted to minimize interference and optimize potential signal in such non-specific analyte capture by, among other methods, choosing release reagents which chemically or enzymatically degrade interfering substances, selecting pre-filters of such a composition that non-specific adsorption of analyte is minimized, and selecting or pre-treating capture surfaces such as to optimize non-specific binding. Since on one hand, non-specific adsorption onto surfaces and pre-filters need to be minimized, and on the other, non-specific binding to the capture surface increased, these methods are often compromising and result in poor potential sensitivities and specificities.
The specificity of an immunoassay is highly dependent upon he efficiency by which an antibody reacts with the analyte in question but not with other similar analytes. If certain potentially interfering organisms demonstrate the ability to bind the antibodies in question by mechanisms not involving the hypervariable region, such as associating with the carbohydrate or Fc regions, screening for non-crossreacting antibodies may become extremely difficult if not impossible.
Other methods of increasing the potential specificity of immunologically based assays include designing competition assays which quantify immunological interactions in an attempt to exclude cross reactants which often, but not always, have a weaker affinity for the antibody in question.